Investigation of the Bacillus cereus phosphonoacetaldehyde hydrolase. Evidence for a Schiff base mechanism and sequence analysis of an active-site peptide containing the catalytic lysine residue

Reaction of Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) with phosphonoacetaldehyde or acetaldehyde in the presence of NaBH4 resulted in complete loss of enzymatic activity. Treatment of phosphonatase with NaBH4 in the absence of substrate or product had no effect on catalysis. Inactivation of phosphonatase with [3H]NaBH4 and phosphonoacetaldehyde, NaBH4 and [14C]acetaldehyde, or NaBH4 and [2-3H]phosphonoacetaldehyde produced in each instance radiolabeled enzyme. The nature of the covalent modification was investigated by digesting the radiolabeled enzyme preparations with trypsin and by separating the tryptic peptides with HPLC. Analysis of the peptide fractions revealed that incorporation of the 3H- or 14C-radiolabel into the protein was reasonably selective for an amino acid residue found in a peptide fragment observed in each of the three trypsin digests. Sequence analysis of the 3H-labeled peptide fragment isolated from the digest of the [2-3H]phosphonoacetaldehyde/NaBH4-treated enzyme identified N epsilon-ethyllysine as the radiolabeled amino acid. The ability of the phosphonatase competitive inhibitor (Ki = 230 +/- 20 microM) acetonylphosphonate to protect the enzyme from phosphonoacetaldehyde/NaBH4-induced inactivation suggested that the reactive lysine residue is located in the enzyme active site. Comparison of the relative effectiveness of phosphonoacetaldehyde and acetaldehyde as phosphonatase inactivators showed that the N-ethyllysine imine that is reduced by the NaBH4 is derived from the corresponding N-(phosphonoethyl) imine. On the basis of these findings, a catalytic mechanism for for phosphonatase is proposed in which phosphonoacetaldehyde is activated for P-C bond cleavage by formation of a Schiff base with an active-site lysine. Accordingly, an N-ethyllsysine enamine rather than the high-energy acetaldehyde enolate anion is displaced from the phosphorus.     

Site of attachment of 11-cis-retinal in bovine rhodopsin

A dipeptide containing the binding site for retinal in bovine rhodopsin has been isolated and its sequence determined. Rhodopsin containing [11-3H]retinal was prepared in chromatographically pure form, and the [3H]retinal was reductively linked to its binding site on opsin by using borane--dimethylamine. The [3H]retinylopsin in octyl glucoside was exhaustively digested with Pronase, and its peptides were separated on silica gel in chloroform/methanol/ammonia [Bownds, D. (1967) Nature (London) 216, 1178--1181] followed by silica gel thin-layer chromatography in two solvent systems. The major retinyl peptide was shown to be alanyl-N epsilon-retinyllysine by amino acid composition, 3H content, and amino acid sequence analysis. The retinyl binding site is located in the carboxyl-terminal region of rhodopsin: when rod cell disk membranes containing [3H]retinal rhodopsin were digested with thermolysin and then reacted with sodium borohydride or borane--dimethylamine, [3H]retinal was reduced onto the F2 (Mr congruent to 6000) fragment, which derives from rhodopsin's carboxyl-terminal region.     

Selective cleavage and anticoagulant activity of a sulfated fucan: stereospecific removal of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity

A linear sulfated fucan with a regular repeating sequence of [3)-alpha-L-Fucp-(2SO4)-(1-->3)-alpha-L-Fucp-(4SO4)-(1-->3)-alpha-L-Fucp-(2,4SO4)-(1-->3)-alpha-L-Fucp-(2SO4)-(1-->]n is an anticoagulant polysaccharide mainly due to thrombin inhibition mediated by heparin cofactor II. No specific enzymatic or chemical method is available for the preparation of tailored oligosaccharides from sulfated fucans. We employ an apparently nonspecific approach to cleave this polysaccharide based on mild hydrolysis with acid. Surprisingly, the linear sulfated fucan was cleaved by mild acid hydrolysis on an ordered sequence. Initially a 2-sulfate ester of the first fucose unit is selectively removed. Thereafter the glycosidic linkage between the nonsulfated fucose residue and the subsequent 4-sulfated residue is preferentially cleaved by acid hydrolysis, forming oligosaccharides with well-defined size. The low-molecular-weight derivatives obtained from the sulfated fucan were employed to determine the requirement for interaction of this polysaccharide with heparin cofactor II and to achieve complete thrombin inhibition. The linear sulfated fucan requires significantly longer chains than mammalian glycosaminoglycans to achieve anticoagulant activity. A slight decrease in the molecular size of the sulfated fucan dramatically reduces its effect on thrombin inactivation mediated by heparin cofactor II. Sulfated fucan with approximately 45 tetrasaccharide repeating units binds to heparin cofactor II but is unable to link efficiently the plasma inhibitor and thrombin. This last effect requires chains with approximately 100 or more tetrasaccharide repeating units. We speculate that the template mechanism may predominate over the allosteric effect in the case of the linear sulfated fucan inactivation of thrombin in the presence of heparin cofactor II.     

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.     

Alkylation of an active-site cysteinyl residue during substrate-dependent inactivation of Escherichia coli S-adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase from Escherichia coli is a member of a small class of enzymes that uses a pyruvoyl prosthetic group. The pyruvoyl group is proposed to form a Schiff base with the substrate and then act as an electron sink facilitating decarboxylation. We have previously shown that once every 6000-7000 turnovers the enzyme undergoes an inactivation that results in a transaminated pyruvoyl group and the formation of an acrolein-like species from the methionine moiety. The acrolein then covalently alkylates the enzyme [Anton, D. L., & Kutny, R. (1987) Biochemistry 26, 6444]. After reduction of the alkylated enzyme with NaBH4, a tryptic peptide with the sequence Ala-Asp-Ile-Glu-Val-Ser-Thr-[S-(3-hydroxypropyl)Cys]-Gly-Val-Ile-Ser-Pro - Leu-Lys was isolated. This corresponds to acrolein alkylation of a cysteine residue in the second tryptic peptide from the NH2 terminal of the alpha-subunit [Anton, D. L., & Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822]. The modified residue derived is from Cys-140 of the proenzyme [Tabor, C. W., & Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040] and lies in the only sequence conserved between rat liver and E. coli S-adenosylmethionine decarboxylase [Pajunen et al. (1988) J. Biol. Chem. 263, 17040-17049]. We suggest that the alkylated Cys residue could have a role in the catalytic mechanism.     

L-myo-inositol 1,4,5,6-tetrakisphosphate is present in both mammalian and avian cells.

When myo-[3H]inositol-prelabelled primary-cultured murine bone-marrow-derived macrophages were challenged with platelet-activating factor (PAF; 200 ng/ml), there was a rapid (2.5-fold at 10 s) rise in the intracellular concentration of D-myo-[3H]inositol 1,4,5-trisphosphate, followed by a rise in myo-[3H]inositol tetrakisphosphate. myo-[3H]Inositol tetrakisphosphate fractions were isolated by high-performance anion-exchange chromatography from myo-[3H]inositol-prelabelled chick erythrocytes and primary-cultured macrophages. In both cases [3H]iditol and [3H]inositol were the only significant products (greater than 90% of recovered radioactivity) after oxidation to completion with periodic acid, reduction with NaBH4 and dephosphorylation with alkaline phosphatase. The presence of [3H]inositol after this procedure is consistent with the occurrence of [3H]inositol 1,3,4,5-tetrakisphosphate in the cell extracts, whereas [3H]iditol could only be derived from D- or L-inositol 1,4,5,6-tetrakisphosphate. When [3H]inositol tetrakisphosphate fractions obtained from (A) unstimulated macrophages, (B) macrophages that had been stimulated with PAF for 40s or (C) chick erythrocytes were subjected to the above procedure, radioactivity was recovered in these polyols in the following proportions: A, 60-90% in iditol, with 10-40% in inositol; B, total radioactivity increased by a factor of 9.8, 94% being recovered in inositol and 8% in iditol; C, 70-80% in iditol and 20-30% in inositol. [3H]Iditol derived from myo-[3H]inositol tetrakisphosphate fractions from macrophages and chick erythrocytes was oxidized to sorbose by L-iditol dehydrogenase (L-iditol:NAD+2-oxidoreductase, 1.1.1.14) at the same rate as authentic L-iditol. D-[14C]Iditol, derived from D-myo-inositol 1,4,5-trisphosphate, was not oxidized by L-iditol dehydrogenase. This result indicates that the [3H]iditol was derived from L-myo-inositol inositol 1,4,5,6-tetrakisphosphate. The data are consistent with rapid PAF-sensitive synthesis of D-myo-[3H]inositol 1,3,4,5-tetrakisphosphate in macrophages, and demonstrate that L-myo-inositol 1,4,5,6-tetrakisphosphate is synthesized in both mammalian and avian cells. The levels of L-myo-[3H]inositol 1,4,5,6-tetrakisphosphate in primary-cultured macrophages are not acutely sensitive to PAF.     

Metabolism of myo-[2-H]Inositol and scyllo-[R-H]Inositol in Ripening Wheat Kernels

Injection of myo-[2-(3)H]inositol or scyllo-[R-(3)H]inositol into the peduncular cavity of wheat stalks about 2 to 4 weeks postanthesis led to rapid translocation into the spike and accumulation of label in developing kernels, especially the bran fraction. With myo-[2-(3)H]inositol, about 50 to 60% of the label was incorporated into high molecular weight cell wall substance in the region of the injection. That portion translocated to the kernels was utilized primarily for cell wall polysaccharide formation and phytate biosynthesis. A small amount was recovered as free myo-inositol and galactinol. When scyllo-[R-(3)H]inositol was supplied, most of the label was translocated into the developing kernels where it accumulated as free scyllo-inositol and O-alpha-d-galactopyranosyl-scyllo-inositol in approximately equal amount. None of the label from scyllo-[R-(3)H]inositol was utilized for either phytate biosynthesis or cell wall polysaccharide formation.     

Characterization of the 5-Hydroxytryptamine Receptor Modulating the Release of 5-[3H]Hydroxytryptamine in Slices of the Human Neocortex

In the rat brain, the presynaptic 5-hydroxytryptamine (5-HT) autoreceptors located on 5-HT terminals correspond to the 5-HT1B subtype. The presence of a 5-HT receptor probably located on 5-HT nerve endings and modulating transmitter release in the human neocortex has been reported, but its detailed pharmacological characterization is not yet available. On the other hand, receptor binding and autoradiographic results indicate that the 5-HT1B receptor subtype is not present in the human brain. We, therefore, studied the modulation of the electrically evoked release of [3H]5-HT by various 5-HT receptor agonists and antagonists in preloaded slices of human neocortex obtained from 18 patients undergoing neurosurgery. The nonselective 5-HT1A/1B/1D receptor agonist 5-carboxamidotryptamine produced a potent inhibition (70% at 0.03 microM) of the electrically evoked release of [3H]5-HT which was blocked by 5-HT receptor antagonists with the following relative order of potency: methiothepin greater than metergoline = methysergide greater than propranolol. The selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin at 0.1 microM did not modify the electrically evoked release of [3H]5-HT. The 5-HT1A/1B receptor agonist RU 24969 was 10 times more potent at inhibiting [3H]5-HT overflow in the rat frontal cortex than in the human neocortex. The potent 5-HT1B receptor antagonist cyanopinodolol did not modify the 5-carboxamidotryptamine-induced inhibition of the electrically evoked release of [3H]5-HT in slices of the human neocortex, but produced by itself a small inhibition of [3H]5-HT overflow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of heparin. Modulation of polysaccharide chain length in a cell-free system.

The formation of heparin-precursor polysaccharide (N-acetylheparosan) was studied with a mouse mastocytoma microsomal fraction. Incubation of this fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded labelled macromolecules that could be depolymerized, apparently to single polysaccharide chains, by alkali treatment, and thus were assumed to be proteoglycans. Label from UDP-[3H]GlcA (approx. 3 microM) is transiently incorporated into microsomal polysaccharide even in the absence of added UDP-GlcNAc, probably owing to the presence of endogenous sugar nucleotide. When the concentration of exogenous UDP-GlcNAc was increased to 25 microM the rate of incorporation of 3H increased and proteoglycans carrying polysaccharide chains with an Mr of approx. 110,000 were produced. Increasing the UDP-GlcNAc concentration to 5 mM led to an approx. 4-fold decrease in the rate of 3H incorporation and a decrease in the Mr of the resulting polysaccharide chains to approx. 6000 (predominant component). When both UDP-GlcA and UDP-GlcNAc were present at high concentrations (5 mM) the rate of polymerization and the polysaccharide chain size were again increased. The results suggest that the inhibition of polymerization observed at grossly different concentrations of the two sugar nucleotides, UDP-GlcA and UDP-GlcNAc, may be due either to interference with the transport of one of these precursors across the Golgi membrane or to competitive inhibition of one of the glycosyltransferases. The maximal rate of chain elongation obtained, under the conditions employed, was about 40 disaccharide units/min. The final length of the polysaccharide chains was directly related to the rate of the polymerization reaction.     

The lipid binding, regulatory domain of protein kinase C. A 32-kDa fragment contains the calcium- and phosphatidylserine-dependent phorbol diester binding activity

Trypsinization of rat brain protein kinase C (80 kDa) into 50- and 32-kDa fragments occurred without inhibition of [3H]phorbol dibutyrate ([3H]PDBu) binding activity. The 50-kDa fragment, the catalytic domain (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616), was further degraded by trypsin, whereas the 32-kDa fragment was resistant. Protein kinase activity and the [3H]PDBu binding activity were completely separated upon gel filtration of a solution containing Triton X-100/phosphatidylserine mixed micelles and trypsinized protein kinase C. Pooled fractions of the [3H]PDBu binding activity contained a 32-kDa fragment exclusively. The binding of [3H]PDBu to this fragment was dependent on calcium and phosphatidylserine and was of high affinity (Kd = 2.8 nM) and of essentially identical specificity to that of native protein kinase C. It is concluded that the 32-kDa fragment represents a lipid binding, regulatory domain of protein kinase C.     

Evidence that forskolin binds to the glucose transporter of human erythrocytes

Binding of [4-3H]cytochalasin B and [12-3H]forskolin to human erythrocyte membranes was measured by a centrifugation method. Glucose-displaceable binding of cytochalasin B was saturable, with KD = 0.11 microM, and maximum binding approximately 550 pmol/mg of protein. Forskolin inhibited the glucose-displaceable binding of cytochalasin B in an apparently competitive manner, with K1 = 3 microM. Glucose-displaceable binding of [12-3H]forskolin was also saturable, with KD = 2.6 microM and maximum binding approximately equal to 400 pmol/mg of protein. The following compounds inhibited binding of [12-3H]forskolin and [4-3H]cytochalasin B equivalently, with relative potencies parallel to their reported affinities for the glucose transport system: cytochalasins A and D, dihydrocytochalasin B, L-rhamnose, L-glucose, D-galactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, phloretin, and phlorizin. A water-soluble derivative of forskolin, 7-hemisuccinyl-7-desacetylforskolin, displaced equivalent amounts of [4-3H]cytochalasin B or [12-3H]forskolin. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either [4-3H]cytochalasin B or [12-3H]forskolin in a glucose-displaceable manner. These results indicate that forskolin, in concentrations routinely employed for stimulation of adenylate cyclase, binds to the glucose transporter. Endogenous ligands with similar specificities could be important modulators of cellular metabolism.     

Chemical and genetic comparison of the glucose and nucleoside transporters

Glucose and nucleoside uptake into human red cells occurs through protein(s) which copurify in a complex, known as band 4.5 of relative mass (Mr) 66,000 to 50,000. The specific inhibitor of glucose transport, [3H]cytochalasin B, and the specific inhibitor of nucleoside transport, [3H]nitrobenzylthioribofuranosylpurine ([3H]NBMPR), incorporate covalently into component(s) of band 4.5 upon irradiation with ultraviolet light. Both photolabelled components are shown to be glycoproteins, since their migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is increased after treatment of photolabelled band 4.5 with endoglycosidase F. Peptide maps of the photolabelled components were compared. Red cell membranes were photolabelled with either [3H]cytochalasin B or [3H]NBMPR and subjected to SDS-PAGE. The region containing band 4.5 was cut and transferred to a second SDS-PAGE system and exposed to either papain or Staphylococcus aureus V8 protease. Papain (5 micrograms) completely cleaved band 4.5 and produced fragments of Mr 33,000, 26,000, 21,000, 15,000, and 12,500. Of these, the 21,000 fragment was the most conspicuous and it retained the label of [3H]cytochalasin B; the 33,000 fragment retained the label of [3H]NBMPR. The V8 protease (0.75 microgram) completely cleaved band 4.5 and produced fragments of Mr 35,000, 28,000, 22,000, 16,000, 13,500, and 9,000. The 28,000 fragment retained the label of [3H]cytochalasin B. The label of [3H]NBMPR was distributed along the gel in several regions comprising the 35,000, 28,000, and 16,000 fragments. Longer treatment with the V8 protease did not alter the position of the 28,000 [3H]cytochalasin B labelled peak, but completely abolished the [3H]NBMPR labelled peaks. Genetic segregation of the glucose and nucleoside transporters was determined in a lymphoma cell line. A mutant (14T- g) of S49 cells was selected which had lost the capacity to transport thymidine or to bind NBMPR. Uptake of either 2-deoxyglucose or 3-O-methylglucose, inhibitable by cytochalasin B, was not impaired in this mutant. It is concluded that the nucleoside and glucose transporters are glycoprotein components of band 4.5, which are differentiated by peptide map analysis. Further, a lymphoblast mutant was isolated which had lost the nucleoside transport function but retained the glucose transport function.     

Ketoconazole inhibits self-induced metabolism of 1,25-dihydroxyvitamin D3 and amplifies 1,25-dihydroxyvitamin D3 receptor up-regulation in rat osteosarcoma cells

Ketoconazole (an inhibitor of vitamin D-24 hydroxylase) was used to study the role of self-induced 1,25-dihydroxyvitamin D3 (1,25-D3) metabolism on cellular responsiveness to 1,25-D3. Eighteen hours of treatment with 1,25-dihydroxy-[26,27-methyl-3H]vitamin D3 (1,25-[3H]D3) increased total 1,25-D3 receptors (VDR) from 60 to 170 fmol mg/protein. In cells treated with both 1,25-[3H]D3 and ketoconazole, up-regulation of VDR was increased by 40% over that observed with cells receiving 1,25-[3H]D3 alone. Ketoconazole alone had no agonistic activity. Treatment of cells with 1 nM 1,25-[3H]D3 plus increasing doses of ketoconazole (0-30 microM) resulted in a dose-dependent increase in occupied VDR and total VDR. This up-regulation was associated with reduced 1,25-[3H]D3 catabolism. 1,25-[3H]D3-induced up-regulation of VDR typically peaked at 14 h and declined thereafter. Ketoconazole lengthened the time to reach peak VDR up-regulation to 20 h. The ability of ketoconazole to increase cell responsiveness (VDR up-regulation) was the result of both increased and prolonged occupancy of VDR by 1,25-[3H]D3. The t1/2 of occupied VDR was 2 h in the absence of ketoconazole and greater than 7 h when ketoconazole was present. Collectively, these results suggested that self-induced catabolism of 1,25-D3 is an important regulator of VDR occupancy and therefore cellular responsiveness to hormone. These data also demonstrate the usefulness of ketoconazole as an inhibitor of vitamin D hydroxylases in intact cells.     

Modulation of [3H]quinpirole binding at striatal D2 dopamine receptors by a monoamine oxidaseA-like site: evidence from radioligand binding studies and D2 receptor- and MAO(A)-deficient mice.

[3H]Quinpirole is a dopamine agonist with high affinity for the D2 and D3 dopamine receptors. A variety of monoamine oxidase inhibitors (MAOIs) inhibit equilibrium binding of [3H]quinpirole binding in rat striatal membranes suggesting that MAOIs interact with a novel binding site that is labeled by [3H]quinpirole or that allosterically modulates [3H]quinpirole binding. To determine whether the D2 receptor is essential for [3H]quinpirole binding and/or modulation of [3H]quinpirole binding by MAOIs, D2 receptor-deficient mice were studied. [3H]Quinpirole binding was decreased in D2 receptor-deficient mice to 3% of that observed in wild-type controls indicating that [3H]quinpirole binding is associated with the D2 dopamine receptors. Then, in an attempt to label the site mediating the modulation of [3H]quinpirole binding, binding of the MAOI [3H]Ro 41-1049 was characterized in rat striatal membranes. [3H]Ro-41-1049 labeled a single binding site with a pharmacological profile with respect to MAOIs that was similar to both [3H]quinpirole binding (Spearman r=0.976) and MAO(A) activity. To determine whether MAO(A) plays a role in the modulation of [3H]quinpirole binding by MAOIs, MAO(A)-deficient mice were examined. In these mice, [3H]Ro-41-1049 binding was decreased to 7% of wild-type control. [3H]Spiperone binding was unaltered. Spiperone-displaceable [3H]quinpirole binding was decreased to 43% of wild-type control; however, the remaining [3H]quinpirole binding in MAO(A)-deficient animals was inhibited by Ro 41-1049 similar to wild-type. [3H]Ro-41-1049 binding was not decreased in D2 receptor-deficient mice. These data suggest that [3H]Ro-41-1049 labels multiple sites and that MAOIs modulate [3H]quinpirole binding to the D2 receptor via interactions at a novel, non-MAO binding site with MAO(A)-like pharmacology.     

Tuftsin-macrophage interaction: specific binding and augmentation of phagocytosis.

The binding of [3H]tuftsin to normal and in vivo stimulated mouse peritoneal macrophage populations was studied at 22 degrees C. The [3H]tuftsin binding to thioglycollate-stimulated macrophages was shown to be rapid and saturable, with an equilibrium dissociation constant (K(D)) (calculated from a Scatchard plot) of 5.3 X 10(-8) M. The calculated number of binding sites per macrophage amounts to approximately 72,000. Binding competition studies with unlabelled tuftsin yielded a K(D) of 5.0 X 10(-8) M. [3H] [N-Acetyl-Thr1]tuftsin, an inactive analog of tuftsin, failed to bind specifically to thioglycollate-stimulated macrophages. [N-Acetyl-Thr1]tuftsin and the tripeptide [Des-Arg4]tuftsin failed to compete for tuftsin binding sites, while [D-Arg4]tuftsin, an analog with small tuftsin-like activity, exhibited a low degree of inhibition of [3H]tuftsin binding. Thus a rather high degree of specificity is involved in the binding of the tetrapeptide. Normal as well as six different macrophage populations induced by stimulation with thioglycollate, concanavalin-A, starch, mineral oil, glucan and Bacillus Calmette Guerrin (BCG), exhibited a similar degree of binding of [3H]tuftsin. Corynebacterium parvum (CP)-stimulated macrophages, on the other hand, showed a 6- to 10-fold-lower capacity for tuftsin binding. Under similar experimental conditions, mouse fibroblast and lymphocyte preparations revealed no detectable specific binding. Tuftsin augmented the phagocytic response of normal and stimulated macrophages assessed both for phagocytosis mediated via the Fc-receptor and via non-specific receptors. CP-stimulated macrophages did not exhibit an increased phagocytic response upon treatment with tuftsin.     

Solubilization of D2 Dopamine Receptor Coupled to Guanine Nucleotide Regulatory Protein from Bovine Striatum

D2 dopamine receptor from bovine striatum was solubilized in a form sensitive to guanine nucleotides, by means of a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). The presence of sodium ion markedly increased the solubilization yield. Treatment of the membranes with 10 mM CHAPS and 0.72 M NaCl solubilized 26% of the stereospecific [3H]spiperone binding sites in the original membrane preparations. The solubilized [3H]spiperone binding sites possessed characteristics of the D2 dopamine receptor: (a) localization of the site in the striatum but not in the cerebellum; (b) high affinity to nanomolar concentrations of [3H]spiperone; (c) displacement of [3H]spiperone binding by nanomolar concentrations of neuroleptics, but only by micromolar concentrations of dopamine and apomorphine; (d) equal activity of various dopamine agonists and antagonists in the soluble and membrane preparations. Guanine nucleotides decreased the affinity of the solubilized D2 dopamine receptor for dopamine agonists, but not for antagonists. The solubilized receptor complex was eluted in Sepharose CL-4B column chromatography as a large molecule, with a Stokes radius of approximately 90 A. These results indicate that the complex between the D2 dopamine receptor and GTP binding protein remains intact throughout the solubilization procedure.     

Synthesis of nucleosides labeled with tritium at the 5'-carbon atom of the ribose residue

Chemical and enzymatic syntheses of [5'-3H]adenosine, [5'-3H]guanosine, and [5'-3H]uridine have been developed. The reduction of beta-D-ribo-pentadialdo-1,4-furanosyl derivatives of corresponding bases is used in the chemical synthesis. The maximum molar activity of the labelled products was 220 TBk/mol in reactions with [3H]NaBH4 and 370-740 TBk/mol in reactions with gaseous tritium. The enzymatic synthesis was performed by the rebosylation of heterocyclic bases with nucleoside phosphorylase and [5'-3H]uridine as a ribosyl donor. Nucleoside phosphorylase is proposed to be used in the immobilized form to avoid the decrease of molar activity. Nucleosides labelled with tritium both in ribosyl and heterocyclic moieties were synthesised enzymatically.     

Synthetic peptide KKRR corresponding to the human ACTH fragment 15-18 is an antagonist of the ACTH receptor

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [3H]ACTH (11-24) and [3H]ACTH (15-18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [3H]ACTH (11-24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (Kd 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) were synthesized, and their ability to inhibit the specific binding of [3H]ACTH (11-24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15-18) (KKRR) (Ki 2.3 +/- 0.2 nM), whose [3H] labeled derivative binds to rat adrenocortical membranes (Kd 2.1 +/- 0.1 nM) with a high affinity. The specific binding of [3H]ACTH-(15-18) was inhibited by 100% by unlabeled ACTH (11-24) (Ki 2.0 +/- 0.1 nM). ACTH (15-18) in the concentration range of 1-1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.     

Changes in serotonin2A and GABA(A) receptors in schizophrenia: studies on the human dorsolateral prefrontal cortex

Having shown a decrease in serotonin2A receptors in the dorsolateral prefrontal cortex (DLPFC) from schizophrenic subjects, we have now determined if this change was reflective of widespread changes in neurochemical markers in DLPFC in schizophrenia. In Brodmann's area (BA) 9 from 19 schizophrenic and 19 control subjects, we confirmed a decrease in the density of [3H]ketanserin binding to serotonin2A receptors in tissue from the schizophrenic subjects [39 +/- 3.3 vs. 60 +/- 3.6 fmol/mg estimated tissue equivalents (ETE); p < 0.005]. In addition, the density of [3H]muscimol binding to GABA(A) receptors was increased in the schizophrenic subjects (526 +/- 19 vs. 444 +/- 28 fmol/mg ETE; p < 0.02). [3H]YM-09151-2, N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine, [3H]SCH 23390, [3H]mazindol, and N(G)-nitro-L-[3H]arginine binding to BA 9 did not differ between groups, and there was no specific binding of [3H]raclopride or 7-hydroxy-2-(di-n-[3H]propylamino)tetralin to BA 9 from either cohort of subjects. This suggests the density of dopamine D1-like and NMDA receptors, the dopamine transporter, and nitric oxide synthase activity are not altered in BA 9 from schizophrenic subjects. The selective nature of the changes in serotonin2A and GABA(A) receptors in DLPFC could indicate that these changes are involved in the pathology of schizophrenia.